Various attempts have been made develop gene therapy for cystic fibrosis (CF) airway disease.
Airway disease is the major cause of morbidity and mortality in cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Welsh et al., The Metabolic and Molecular Basisof Inherited Disease, eds. Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Childs, B. & Vogelstein, B. (McGraw-Hill, New York). Gene transfer offers the potential for a new and effective treatment for CF airway disease. For reviews see Davies, Geddes & Alton, 2001, J. Gene Med. 3:409-417; Flotte, 1999, Curr. Opin Mol. Ther. 1:510-516; and Welsh, 1999, J. Clin. Invest. 104:1165-1166. Previous studies have shown the feasibility of transferring the CFTR cDNA to CF airway epithelial cells in vitro and in vivo. However, with most vectors two main problems limit gene transfer: gene transfer from the apical surface of differentiated airway epithelia is inefficient, and DNA molecule expression is transient. See Davies, Geddes & Alton, 2001, J. Gene Med. 3:409-417; Flotte, 1999, Curr. Opin Mol. Ther. 1:510-516; and Welsh, 1999, J. Clin. Invest. 104:1165-1166.
For developing CF gene therapy, adeno-associated virus (AAV) vectors have several potential advantages.
One limitation of AAV vectors is the small size of a DNA molecule that can be inserted. Studies testing the insert size suggest that 4100-4900 bp is the optimal genome size for packaging. See Dong, Fran & Frizzell, 1996, Hum Gene Ther. 7:2101-2112. In comparison, the coding sequence of full length CFTR is 4450 bp. Riordan et al., 1989, Science 245:1066-1073. Addition of the two inverted terminal repeats of AAV (300 bp), and minimal 3′ and 5′ untranslated regions (˜100 bp) yields an insert (4850 bp) that leaves little room for promoter-enhancer elements, most of which are >600 bp. Some studies have attempted to circumvent this limitation by using AAV sequences as a promoter. See Zhang et al., 1998, Proc. Natl. Acad. Sci. 95:10158-10163; and Flotte et al., 1993, J. Biol. Chem. 268:3781-3790. However, their utility in differentiated airway epithelia and in vivo is uncertain.
A potential solution to this problem is to shorten the DNA molecule by selectively deleting coding sequence. This strategy has been proposed with a mini-dystrophin gene for Duschennes muscular dystrophy (Phelps et al., 1995, Hum. Mol. Genet. 4:1251-1258) and for CFTR (Zhang et al., 1998, Proc. Natl. Acad. Sci. 95:10158-10163; and Flotte et al., 1993, J. Biol. Chem. 268:3781-3790).
The CFTR R (regulatory) domain (for reviews on the R domain see Ostedgaard, Baldursson & Welsh, 2001, J. Biol. Chem. 276:7689-7692; Sheppard & Welsh, 1999, Physiol. Rev. 79:S23-S45; Gadsby & Nairn, 1999, Pysiol. Rev. 79:S77-S107; and Ma, 2000, News Physiol. Sci. 15:154-158) has been speculated to be an important domain. Earlier studies in heterologous cells indicated that the CFTR R domain is predominantly random coil and that parts of the R domain can be deleted without abolishing channel function. Phosphorylation of the R domain by the cAMP-dependent protein kinase (PKA) controls CFTR Cl− channel activity. Although this domain contains several conserved serines that are phosphorylated by PKA, no one phosphoserine is required and several different phosphoserines contribute to regulation. While the boundaries of the R domain are not precisely defined, they extend approximately from residues 634-708 at the N-terminus to approximately 835 at the C-terminus. See Ostedgaard, Baldursson & Welsh, 2001, J. Biol. Chem. 276:7689-7692; Ostedgaard, et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97:5657-5662; and Csandy et al., 2000, J. Gen. Physiol. 116:477-500. Previous work has shown that residues 708-831 regulate activity, but in solution they are predominantly random coil. Ostedgaard, et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97:5657-5662. These studies suggest that selective deletions might not severely disrupt structure and that retention of consensus phosphorylation sites might be sufficient for PKA-dependent regulation. Importantly, several earlier studies deleted portions of the R domain without abolishing channel function. Zhang et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:10158-10163; Rich et al., 1991, Science 253:205-207; Rich et al., 1993, Receptors Channels 1:221-232; Ma et al., 1997, J. Biol. Chem. 272:28133-28141; Vankeerberghen et al., 1999, Biochemistry 38:14988-14998; and Xie et al., 2000, Biophys. J. 78:1293-1305.
While these earlier studies suggested that a DNA molecule with R domain deletions might be of value in gene therapy applications, some alterations induced channel activity in the absence of phosphorylation, reduced the response to PKA-dependent phosphorylation, and/or reduced net channel activity. Zhang et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:10158-10163; Ostedgaard, Baldursson & Welsh, 2001, J. Biol. Chem., 276:7689-7692; Rich et al., 1991, Science 253:205-207; Rich et al., 1993, Receptors Channels 1:221-232; Ma et al., 1997, J. Biol. Chem. 272:28133-28141; Vankeerberghen et al., 1999, Biochemistry 38:14988-14998; and Xie et al., 2000, Biophys. J. 78:1293-1305. Moreover, previous studies have only examined CFTR expressed in heterologous cell lines and studied activity using the patch-clamp technique, planar lipid bilayers, or anion efflux. There is no information, prior to this invention, about their function in airway or other epithelia. Expression in epithelia is key in assessing their value for gene transfer because deletions could alter protein-protein interactions, targeting to the apical membrane, constitutive and stimulated activity, phosphorylation-dependent regulation, and perhaps toxicity.
The present invention solves these problems by deleting regions within the CFTR R (regulatory) domain (for reviews on the R domain see Ostedgaard, Baldursson & Welsh, 2001, J. Biol. Chem. 276:7689-7692; Sheppard & Welsh, 1999, Physiol. Rev. 79:S23-S45; Gadsby & Nairn, 1999, Pysiol. Rev. 79:S77-S107; and Ma, 2000, News Physiol. Sci. 15:154-158) to provide a partially deleted CFTR capable of forming Cl− channels in airway epithelia in vitro and in vivo.